Method for determining female serum cytotoxicity to male monoclear cells

FIELD: medicine.

SUBSTANCE: method involves determining female serum cytotoxicity to male lymphocytes, including a combined culture with reference male and analysed female serum in a 96-well tray in the presence of the nutrient medium RPMI 1640 in a CO2 incubator. One day later, lymphocytes are counted in the well in a Goryaev's chamber with the male (reference) and female (analysed) serum. That is followed by determining a cytotoxic index (CI), which represents a quotient of the analysed cell count and the reference cell count. The normal cytotoxic index makes approximately 0.7 and less.

EFFECT: invention enables studying the responses of female humoral immune factors to male antigens and evaluating a risk of miscarriage, early spontaneous abortions and missed miscarriages.

1 ex

 

The increase in the number of infertile marriages registered in Russia and other countries, dictates the need to find modern methods of diagnosis and treatment of infertility, especially in cases where the reason it is not installed. One of the causes of infertility can be a high degree of tissue compatibility of the spouses and, as a consequence, a weak immune response and low production of protective factors. As a result of an immune response to the antigens responsible for the generation of specific cytotoxic antibodies against antigens of the father. This is a blocking antibody IgG presents. They bind to Fc receptors on lymphocytes (NK-cells) and, thus, prevent the accumulation of activated NK cells with high cytotoxic activity.

It is shown that this mechanism works most efficiently when the maximum differences of the spouses to the antigens of major histocompatibility complex (HLA antigens). Weak immune response and insufficient production of protective factors leads to the development of early gestational complications - spontaneous miscarriage or missed abortion.

As a result of an immune response to HLA antigens are produced by specific protective factors: cytotoxic antibodies against antigens of the father (ARSA - antipaternal cytotoxic antibodies); Ab2 anti-anti HLA antibodies (Ab2-anti-anti HLA antibodies); antibodies that block mixed cult�ru lymphocytes (MLR-Bf - mixed lymphocyte reaction blocking factor) [1, 2].

Laboratory tests to confirm the diagnosis alloimmune nature of miscarriage include HLA typing, the definition of maternal lymphocytotoxicity antibodies against the father, a blocking antibody to a mixed culture of cells (SCR), etc.

That these laboratory studies may also be useful in the diagnosis of infertility, demonstrated by the fact that 85% of pregnancies are terminated before the fact of their establishment. It is assumed that recurrent pregnancy loss and infertility are manifestations of existing immune disorders [3-6].

The invention relates to medicine, in particular to methods of determining the state of the immune system, and relates to a method of determining cytotoxicity of the serum female to male lymphocytes.

Analogous to the claimed method is a Method for quantifying the concentration of antibodies specific to the antigen endometrial stromal cells human tissue, in biological fluids of a person, containing specific antibodies" [10].

Method is to use the traditional enzyme-linked immunosorbent assay (ELISA) and includes chemical binding of the antigen with the surface of the microplate, followed by biological fluid containing antibodies, micro�Lancet, incubation, carrying out the color reaction and spectrophotometric evaluation of indicators of the color reaction. This produces a target antigen, exposing the enzymatic hydrolysis of the samples of normal human tissues containing stromal cells of the endometrium. The enzymatic hydrolysis reaction is stopped by addition of a chelating agent. Received a mixed cell suspension is subjected to centrifugation in a continuous density gradient, resulting in isolated stromal cells. Microsomal fraction of stromal cells used as target antigen in the selected dilutions covalently associated with the surface of the microplate wells, then to make a series of holes in predetermined dilutions of the calibration material, prepared on the basis of the standard drug is specific to the target antigen of antibodies, in another series of holes - measured sample of a biological liquid and once the traditional TYPHUS by spectrophotometric indices build a calibration curve and determine the concentration of specific antibodies.

The disadvantage of analog is the complexity of technology the cells of normal human tissues to identify the antigen, proper calibration, obtaining the necessary components and equipment setting reaction.

As� prototype, the authors propose a Method of determining the blocking effect of autologous serum [11].

The method consists in the fact that the lymphocytes were incubated in medium with addition of phytohemagglutinin, in the environment with the addition of phytohemagglutinin and autologous serum in the medium with the addition of phytohemagglutinin and polerowanej AB-serum and in the absence of additives, followed by the addition of FITC-labeled antibodies to CD69. Further assessment of the fluorescence in each sample and determine the inhibitory effect on the proposed formula.

The disadvantages of the prototype are the complexity of implementation, expensive materials and equipment, lack of trained this technique personnel.

The problem to be solved by the claimed invention is to simplify the methodology for the determination of antibodies (blocking factors) serum female to male lymphocytes, reducing the time of carrying out the method and to assess the possible risk of miscarriage.

This problem is solved due to the fact that the claimed method of determining the cytotoxicity of the serum female to male lymphocytes can be performed proposed by the authors in the following manner.

A collection of blood in women is 10-20 day cycle. The blood in an amount of 5 ml is taken from women in a single tube blood collection tubes with red cap on the serum. In men taking blood 2 vials 5 ml: one with a red cover on the serum, the other with a green tube with �eparina for isolation of lymphocytes.

Tubes of blood with red cap mark put in a separate stand for 10 min in a thermostat at 37°C (at room temperature 20 min), then 20 min in the fridge. Then centrifugum 3 thousand rpm for 10 min. Serum was collected in a sterile tube.

For receiving male blood lymphocytes layered on ficol-verografin (density 1.077) (LTD. Company Panaco" Moscow) and centrifugum 20 min at 3 thousand rpm the resulting interphase lymphocytes washed three times, first in buffered physical solution (0.01 M, pH=7,4), then in a nutrient medium RPMI 1640. Determine the number of cells in 1 ml by count in the camera Goryaeva and calculates the concentration so that 1 ml was 2 million lymphocytes.

In 96-well sterile tablet insertion in the 1st and 2nd hole 100 µl of cell suspension, 20 μl of RPMI medium 1640, 1 insertion hole 30 ál serum women (experience), the 2nd - 30 ál serum men (control), resuspended and set for 24 hours at 37°C in CO2an incubator.

A day believe the number of lymphocytes in the presence of female Sivaratri (experience) and male (control).

The cytotoxic index expressing the number obtained as a result of the private from dividing the number of cells in the experiment on the number of lymphocytes in the control. Normal cytotoxic index equal to about 0.7 and below.

It �the means, in women's serum contains factors that inhibit the proliferation of male lymphocytes in pregnancy and there will be no attack of the killer cells to the antigens of the fetus.

The technical result provided by the above combination of features is the reliability, efficiency, speed of execution and efficiency of determining the level of antibodies (blocking factors) serum female to male lymphocytes, speeding up the diagnosis.

The proposed method will provide a fast, a day from the beginning of studies, economical and efficient to undertake a study of the reaction of humoral factors of the immune system of the female body to male antigens and to assess the risk of miscarriage, early pregnancy loss, non-developing pregnancy, etc.

The method is illustrated by the following example.

We examined 36 women who had pregnancy and childbirth proceeded without physiological abnormalities, 32 women with recurrent miscarriage of unknown origin.

The cytotoxic index of 87% women, the first group was 0,7±0,2, 13% - 0,5±0,26.

The cytotoxic index in 79% of women of the second group with recurrent miscarriage of unknown origin was 2,5±0,7, 21% - 1,0±0,6.

Method for determining cytotoxicity of the serum female to male lymphocytes will allow to assess the reaction of the immune system women�s to male antigens and predict possible risks of miscarriage. This study gives the opportunity to take timely corrective action by assigning therapeutic interventions. To them currently include the conduct of alloimmunization (Permission to use FS No. 2009/179 from 2.07.2009 G. ) and the use of the drug normal immunoglobulin for intravenous administration [7-9].

Thus, the proposed method will enable rapid diagnosis of immune disorders in women, and as a result will increase the birth rate.

Bibliography:

1. Pandey M. K., Agrawal S. Induction of MRL-Bf and protection of fetal loss: a current double blind randomized trial of paternal lymphocyte immunization for women with raccurent spontaneous abortion. Int Immunopharmacol 2004; 4:289-298.

2. Ito K., Tanaka T., Tsutsumi N. Possible mechanisms of immunotherapy for maintaining pregnancy in recurrent spontaneous aborters: analysis of antiidiotype antibodies directed against autologous T-cell receptors. Hum Reprod 1999; 14:650-655.

3. Bezkorovainy T. C, I. V. Poltavets, Twin E. A., Wasserman N. N., Tverskaya CM., Polyakov L. V. the Role of class II antigens of major histocompatibility complex in habitual miscarriage. Problems of reproduction 2006; 12:2.46-54.

4. Khonina N. And., Tikhonova M. A., Dzutseva I. B., Pasman N. M. Ostanin A. A., Chernykh E. R. Immune dysfunction in women with unexplained infertility. Honey. immunology 2010; 12:6:511-520.

5. Yamada N., Atsumi, T., Kato, E. N. Prevalence of diverse antiphospholipid antibodies in women with recurrent spontaneous abortion. Fertil Steril 2003; 80:1276-1278.

6. Koyama M., Saji F., Takahashi S., Takemura M. Probabilistic assessmen of the HLA-sharing of recurrent spontaneous abortion couples in Japanese population. Tissue antigen 2010; 37:211-217.

7. Chernyshov V. N., Sudoma I. A., don, B. V., Kostiuczyk A. A., Masliy Y. the Value of the increased cytotoxicity of natural killer cells during repeated failure of in vitro fertilization. Phys. The Academy of medical Sciences of Ukraine, 2010, vol. 16, No. 2, pp. 288-298.

8. Markov S. A., Men'shikov V. I., Bedulev L. V., ] N.N., Kazakov I. G., Veretennikova K. G. Clinical and diagnostic value of metocolopramide immune response of the mother to the antigens of the father and of the establishment of tolerance in pregnancy. Bulletin of Udmurt University. 2011. Vol. 4. Biology, earth science, Physiological studies, pp. 102-106.

9. Khonina N. A., Broitman E. V., Shevela E. Y., Pasman N. M., Chernykh E. R.. Mixed lymptocyte reaction blocking factors (MRL-bf) as potential biomarker for indication and efficacy of paternal lymphocyte immunization in recurrent spontaneous abortion//Arch. Cynecol. Obstet-2013. - Vol. 288. - P. 933-937.

10. Mihnina E. A., Komarov E. K., Khokhlov p. P. Method for the quantitative determination of the concentration of antibodies specific to the antigen endometrial stromal cells human tissue, in biological fluids of a person, containing specific antibodies. Patent No. 2303267 dated 08.06.2005.

11. Sukhikh G. T., Vanko L. V., Sidelnikov V. M., Nikolaeva M. A., Ziganshin M. M., Krechetova L. V. Method of determination of the blocking effect of autologous serum. Patent No. 2396566 from 29.04.2009.

Method for determining cytotoxicity of the serum female to male lymphocytes, including their together�Noah cultivation with control male and investigated female serum in the wells of 96-well plates in the presence of a nutrient medium RPMI 1640 in the CO 2incubator during the day, followed by counting the number of lymphocytes in the cell Goryaeva and determination of cytotoxic index, expressed in private from dividing the number of cells in the experiment on the number of cells in the control at the normal rate of about 0.7 and below.



 

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